Single-strand conformation polymorphism (SSCP) analysis of the molecular pathology of hemophilia B

Abstract

In the present study, we report the application of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis to the screening of seven functionally important factor IX gene (FIX) regions (total length 2.66 kb) in 9 unrelated haemophilia B patients of Portuguese or African origin. In eight of the patients an altered migration pattern of single-stranded DNA was observed. Direct sequencing of the relevant DNA fragments unveiled the following sequence alterations: two novel mutations, namely FIXBarcelos Thr-380-Pro and FIXLousada 9bp insertion at position 31,309 or 31,318; five mutations previously reported in other ethnic groups (FIXPorto Arg-145-His, FIXLuanda Gly-207-Arg, FIXPenafiel Arg-248-Gln, FIXSesimbra Arg-333-Gln, FIXCascais Arg-333-Stop); and a normal variant, G-->T transvertion at position 6,596 in intron 2. We propose hypothetical models for the generation of the 9 bp duplication (FIXLousada). We have performed molecular modeling studies in order to predict the structure of the variant FIX molecules.

Five novel factor IX mutations in unrelated hemophilia B patients

“…The G -82 to T base substitution in intron 2, already described by us (David et al, 1993), was found in a second patient of African origin. Allele-specific amplification analysis of 162 independent X chromosomes from Portuguese (98) and African black (64) populations, allowed us to establish the frequency of the dimorphic T allele as 4.7%, unique to the African black population.…”

“…DNA preparation, amplification by polymerase chain reaction (PCR), SSCP analysis, and direct sequencing were carried out essentially as described by David et al (1993David et al ( , 1994. (Annealing temperatures for the co-amplification of different fragments and oligonucleotides used are available from the authors on request.)…”

Molecular Genetic Analysis of Factor XI Deficiency: Identification of Five Novel Gene Alterations and the Origin of Type II Mutation in Portuguese Families

“…The formation of a Moebius loop-like structure during DNA replication may serve as an intermediate which potentiates the insertion. We have observed a similar sequence structure in the case of a 9 bp insertion in the FIX gene (28). Alternatively, a mechanism based on slipped mispairing and resynthesis of the 7 bp sequence could also explain this duplication.…”

Female haemophiliac homozygous for the factor VIII intron 22 inversion mutation, with transcriptional inactivation of one of the factor VIII alleles

“…Segregation of X chromosomes were studied using polymorphisms in the FIX ( Hin fI in intron A and Mnl I in exon VI) and FVIII ( Bcl I in intron 18, Xba I in intron 22) genes and at the DXS52 locus ( Bcl I) by Southern blot analysis or polymerase chain reaction (PCR), performed as previously described [13,14].…”